Induction of Angiopoietin-2 gene expression by COX-2: A novel role for COX-2 inhibitors during hepatocarcinogenesis Shinji Tanaka, Jack R. Wands, Shigeki.

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Induction of Angiopoietin-2 gene expression by COX-2: A novel role for COX-2 inhibitors during hepatocarcinogenesis Shinji Tanaka, Jack R. Wands, Shigeki Arii Journal of Hepatology Volume 44, Issue 1, Pages 233-235 (January 2006) DOI: 10.1016/j.jhep.2005.09.012 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 In vitro studies of human Hep3B HCC cells (A,B) and in vivo analysis of murine HCC cells (MH134) in C3H mice (C–E). (A) Gene expression of Ang-2 was analyzed by RT-PCR in Hep3B cells transiently transfected with COX-2 expression plasmid (0.1, 1μg) or mock plasmid (control). The COX-2 expression plasmid was kindly provided by Dr Timothy Hla (University of Connecticut). Equal expression of beta-actin demonstrated the integrity of the mRNA in each sample. (B) RT-PCR analysis of Ang-2 expression in Hep3B cells following a 1hr-treatment of interleukin (IL)-1beta, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, tumor growth factor (TGF)-beta, or prostaglandin (PG) E2 (5μM). Equal expression of beta-actin demonstrated the integrity of the mRNA in each sample. (C) In vivo expression of Ang-2 was analyzed by RT-PCR in a murine model of HCC. In brief, 2.5×104 MSH134 cells were inoculated subcutaneously in the flank of 6-week old syngeneic female C3H mice. Subcutaneous tumors (0.5mm3) developed 7-days post-inoculation and the mice were randomized to either control group (n=5) or the NS-398 treatment group (n=5). The treated mice were given NS-398 10mg/kg (body weight) intraperitoneally twice daily for 14 days. Tumor size was observed daily, and mice were sacrificed on day 14 for examination of Ang-2 expression in tumor tissue. Equal expression of beta-actin demonstrated the integrity of the mRNA in each sample. (D) Expression of CD31 protein in the endothelium of vessels surrounding and penetrating a mouse HCC tumor. Sections of a MH134 tumor tissue were analyzed by immunohistochemistry using the anti-CD31 antibody (x400). Note the lack of new vessel formation in a representative example of tumor treated with NS398 compared to a control. (E) Tumor growth rate in the control and NS-398 treated group in a murine model using C3H-derived MH134 HCC cells. The volume of MH134 tumors was determined every two days following treatment. While little effect of NS-398 was observed on MH134 cell proliferation or apoptosis in vitro (data not shown), there was significant inhibition of HCC growth in vivo presumably as the result of its anti-angiogenic properties. Journal of Hepatology 2006 44, 233-235DOI: (10.1016/j.jhep.2005.09.012) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions